The Incidence of ANA and ETI-dsDNA Detected by Enzyme Immunoassays and Indirect Immunofluorescence Assay (IFA)

Abstract

While the SL E (Systemic Lupus Erythematosus) specificity of ANA is low, that of anti-dsDNA autoantibodies is high. The DNA used in the assay must be double stranded: autoantibodies to single -stranded (ss) DNA exist in many diseases and specific to none. The prevalence (70%) of antidsDNA autoantibodies is much higher in SL E, giving a higher diagnostic sensitivity than the similarly disease-specific anti-Sm autoantibodies (30%). Anti-dsDNA autoantibodies are usually detected by very analytically sensitive techniques, such as ELISA (Enzyme Linked Immunosorbent Assay). Within SLE, ds-DNA autoantibodies tend to associate with the presence of glomerulonephritis. Their levels are used to monitor disease activity. We suggest the use of ds-DNA to find the difference between SL E patients with benign variants and classical syndrome of severe skin and renal disease.

The Incidence of ANA and ETI-dsDNA Detected by Enzyme Immunoassays and Indirect Immunofluorescence Assay (IFA)

Izeta Aganovic-Musinovic1, Lama Prljaca-Zecevic2, Djemo Subasic2

1Center for Genetics, Faculty of Medicine, University of Sarajevo, Bosnia and Herzegovina

2Department for Clinical Immunology, Clinical Center of University of Sarajevo, Bosnia and Herzegovina

original papersUMMarYWhile the slE (systemic lupus Erythematosus) specificity of AnA is low, that of anti-dsDnA autoantibodies is high. The DnA used in the assay must be double stranded: autoantibodies to single stranded (ss) DnA exist in many diseases and specic to none. The prevalence (70%) of anti-dsDnA autoantibodies is much higher in slE, giving a higher diagnostic sensitivity than the similarly disease-specic anti-sm autoantibodies (30%).Anti-dsDnA autoantibodies are usually detected by very analytically sensitive techniques, such

as ElisA (Enzyme linked immunosorbent Assay).

Within slE, ds-DnA autoantibodies tend to associate with the presence of glomerulonephritis. Their levels are used to monitor disease activity. We suggest the use of ds-DnA to nd the dierence between slE patients with benign variants and classical syndrome of severe skin and renal disease.

Keywords: sle, systemic lupus erythematosus, ana, anti-nuclear antibodies, ds-dna, double-stranded dna antibodies, IFa, immunoourescence assay, elIsa, enzyme linked immunosor-bent assay

corresponding author: Izeta aganovic, Md. center for Genetics, Faculty of Medicine, university of sarajevo, cekalusa 80, 71000 sarajevo, Bosnia and herzegovina

1. IntroductIon

The sera-immunological hallmark of SLE is anti-nuclear antibody (ANA). In the absence of ANA, the diagnosis of SLE is put into question. So, the ANA is very sensitive test for SLE, being present in virtually all patients and frequently at high titers. Its disease specificity is relatively low since it is frequently found in other rheumatic diseases, as well as in autoimmune liver disease, during viral infection, and , occasion-ally, at low titers, in normal subjects. It has tendency to increase in prevalence with age in healthy adults.

While the SLE (Systemic Lupus Erythematosus) specicity of ANA is low, that of anti-dsDNA autoantibodies is high. The DNA used in the assay must be double stranded: autoantibodies to single stranded (ss) DNA exist in many diseases and specic to none

(1). The prevalence (70%) of anti-dsDNA autoantibodies is much higher in SLE, giving a higher diagnostic sensitivity than the similarly disease-specic anti-Sm autoantibodies (30%). Anti-dsDNA autoantibodies are usually detected by very analytically sensitive techniques, such as ELISA (Enzyme linked immunosorbent assay) (1). Within SLE, ds-DNA autoantibodies tend to associate with the presence of glomerulonephritis. Their levels are used to monitor disease activity.

2. MaterIal and Methods

During the period of a year (January 2008-January 2009), we have analyzed 2132 serum specimens using IF method and 1188 sera specimens using ELISA method for ANA and ds-DNA respectively. The most commonly used method for ANA testing is Im-

munouorescence Assay (2, 3). The basic principle of the procedure is using slides with epithelial cells (Hep-2 cells) as substrate that is incubated in few steps with diluted serum. The unbound material is removed by aspirating and washing. The drop of the uorescence conjugate (anti human IgG uorescein labeled containing Blue dye and 0.099 sodium azid) is added (4). Depending on the amounts of autoantibodies in specimens, using IF microscope, it is possible to detect dierent intensity degree of apple-green uorescence light. Fluorescence grade is determined as: 4+; 3+; 2+ and +. The ELISA kits are solid-phase enzyme immunoassays. Antigen-precoated microplate wells are incubated with calibrators, controls and serum specimens. During the incubation, antibody present in the test sample binds to the coated wells. Horseradish peroxides-conjugated anti-human IgG is incubated in the wells to recognize the autoantibodies bound to the coated wells. Chromogen is added and auto-antibodies are measured using a spectrophotometer plate reader. At end of each incubation, the unbound material is removed by aspirating and washing. When ANA is considered the positive result value is above 23 while the positive result value for ds-DNA is above 60. Results in range of 20 to 40 are considered negative, while results in range value of 40 to 60 are considered limited positive.

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The incidence of AnA and ETi-dsDnA Detected by Enzyme immunoassays and indirect immunouorescence Assay (iFA)

ANA IFA

3%

1%

9%

13%

3. results

During the period of a year we have analyzed 2132 serum specimens using IF method. 924 specimens have been analyzed for ANA antibodies, while 1208 specimens have been analyzed for ds-DNA. All results were interpreted using determined uorescence grade as 4+; 3+; 2+; + and negative.

Among those 924 specimens analyzed for ANA antibodies the following result were obtained: 74% (683) specimens were negative

13% (118) specimens were + 9% (84) specimens were 2+ 3% (29) specimens were 3+ 1% (10) specimens were 4+. Results are presented on Graph 1. The same IF method has been used

for ds-DNA detection. 1208 specimens were analyzed for ds-DNA antibodies and following results were obtained: 94% (1131) specimens were negative

2% (25) specimens were + 3% (42) specimens were 2+ 1% (10) specimens were 3+. Results are presented on Graph 2. During the same period 1188 serum

specimens were analyzed using ELISA method. 745 serum specimens were analyzed for ANA antibodies and results were interpreted as negative if <23 and positive if >23. Results obtained were:

58% (435) specimens were negative 42% (310) specimens were positive Results are presented on Graph 3. For ds-DNA using ELISA method were analyzed 443 serum specimens. Results in range of 20 to 40 are considered negative, while results in range value of 40 to 60 are considered limited positive. Results above 60 were positive.

Results obtained were: 68% (299) specimens were negative

9% (41) specimens were limited positive

23% (103) specimens were positive Results are presented on Graph 4.

4. dIscussIon

Immunouorescence assay of ANA showed 74% negative results and 23% positive, either 2+, 3+ or 4+. ELISA tests for ANA antibodies showed 58% negative results and 42% positive results that in comparing with results achieved using IF ANA seems to be large number of positive patients, but consider-

ing practicing and quality of ELISA it could be expected. Some studies demonstrated that the commercially available ANA-EIA kits show dierent levels of sensitivity and specicity (4, 5). On the other hand, at increased temperatures dsDNA denaturizes into ss-DNA that results in false negative results using IF method (6, 7). That is the reason why ELISA should be used as conrmation method in those cases. Positive ANA test is not specic for SLE and can be associated with many illnesses as: RA, Sjorgens syndrome, scleroderma and infectious diseases as mononucleosis, autoimmune thyroid and liver disease. Furthermore, certain medications can cause positive ANA; many healthy people with no expressed illness have a positive ANA (4, 8).

Antibodies to dsDNA (anti-ds-DNA, dsDNA-Ab) are frequently found in systemic lupus erythematosus, especially during active disease and dier with respect to immunoglobulin classes and avidity (9). Immunofluorescence assay of dsDNA showed 94% negative specimens and 6% of positive specimens either +, 2+ or 3+. Using ELISA method 68% of specimens were negative, 9% were bordered positive and 23% were positive. Detection of anti-dsDNA may precede the diagnosis of SLE by more than a year. Fluctuations in the level of anti-ds-DNA in an individual patient may give important information on the clinical status of the patient (9).

Most of commercial ELISA test systems have great advantages in routine laboratory testing but often detect dsDNA-Ab which are not specic for SLE and therefore give false positive results for non-SLE patients (10, 11).

Anti-dsDNA antibody test incorporated in criteria for the classication of SLE needs updating to reect current insights and technical achievements, including allowance for the presence of non-pathological anti-dsDNA antibodies (12). Anti-DNA of low avidity occurs in rheumatic diseases other than SLE as well, making detection of such antibodies of less diagnostic value (13). It has often been tried to discriminate between clinical subsets of this heterogeneous disease by studying dierences within the population of anti-dsDNA antibodies (14). Immunospecicity, complement-xing ability, avidity, immuno-

globulin (sub) class composition have all been the subject of dierent studies, yet conclusions are contradictory and it has not been elucidated (14, 15).

5. conclusIon

The most precise method used for SLE detection and keeping up with petition status is ELISA anti-dsDNA, still needs updating. Furthermore, in the presence of antibodies ANA should no longer be considered a valid criterion.

This strategy might ultimately facilitate the dierence between SLE patients with benign disease variants and classical syndrome of severe skin and renal disease in pathogenic anti-ds-DNA antibodies.

ANA IFA

3%

1%

9%

13%

74%

negative + ++ +++ ++++

Graph 1. DETECTION OF ANA-ANTIBODIES USING IFA METHOD

The same IF method has been used for ds-DNA detection. 1208 specimens were analyzed for ds-DNA antibodies and following results were obtained:- 94% (1131) specimens were negative- 2% (25) specimens were +- 3% (42) specimens were 2+- 1% (10) specimens were 3+.

Results are presented on Graph 2.

ds - DNA IFA

++ 3%

negative + ++ +++ ++++

Graph 1. DETECTION OF ANA-ANTIBODIES USING IFA METHOD

The same IF method has been used for ds-DNA detection. 1208 specimens were analyzed for ds-DNA antibodies and following results were obtained:- 94% (1131) specimens were negative- 2% (25) specimens were +- 3% (42) specimens were 2+- 1% (10) specimens were 3+.

Results are presented on Graph 2.

ds - DNA IFA

++ 3%

74%

Graph 1. Detection of AnA-antibodies using iFA method

+++

1%

+ 2%

+++

1%

negative 94%

+ 2%

ANA ELISA

negative + ++ +++

Graph 2. DETECTION OF ds-DNA USING IFA METHOD

During the same period 1188 serum specimens were analyzed using ELISA method. 745 serum specimens were analyzed for ANA antibodies and results were interpreted as negative if <23 and positive if >23. Results obtained were:- 58% (435) specimens were negative- 42% (310) specimens were positive

Results are presented on Graph 3.

negative 94%

Graph 2. Detection of Ds-DnA using iFA method

42%

negative + ++ +++

Graph 2. DETECTION OF ds-DNA USING IFA METHOD

During the same period 1188 serum specimens were analyzed using ELISA method. 745 serum specimens were analyzed for ANA antibodies and results were interpreted as negative if <23 and positive if >23. Results obtained were:- 58% (435) specimens were negative- 42% (310) specimens were positive

Results are presented on Graph 3.

ANA ELISA

58%

42%

3

0 - 23, negative > 23, positive

Graph 3. DETECTION OF ANA-ANTIBODIES USING ELISA METHOD

For ds-DNA using ELISA method were analyzed 443 serum specimens. Results in range of 20 to 40 are considered negative, while results in range value of 40 to 60 are considered limited positive. Results above 60 were positive.

Results obtained were:- 68% (299) specimens were negative- 9% (41) specimens were limited positive - 23% (103) specimens were positive

Results are presented on Graph 4.

ds - DNA- ELISA

9%

3

58%

0 - 23, negative > 23, positive

Graph 3. DETECTION OF ANA-ANTIBODIES USING ELISA METHOD

For ds-DNA using ELISA method were analyzed 443 serum specimens. Results in range of 20 to 40 are considered negative, while results in range value of 40 to 60 are considered limited positive. Results above 60 were positive.

Results obtained were:- 68% (299) specimens were negative- 9% (41) specimens were limited positive - 23% (103) specimens were positive

Results are presented on Graph 4.

ds - DNA- ELISA

9%

Graph 3. Detection of AnA-antibodies using ElisA method

4

23%

68%

23%

0 - 40, negative 40- 60, border positive >60, positive

Graph 4. DETECTION OF DS-DNA ANTIBODIES USING ELISA METHOD

4. Discussion

Graph 4. Detection of Ds-DnA antibodies using ElisA method

68%

0 - 40, negative 40- 60, border positive >60, positive

Graph 4. DETECTION OF DS-DNA ANTIBODIES USING ELISA METHOD

4. Discussion

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MED ARH 2010; 64(2) ORiginAl pApERs

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The incidence of AnA and ETi-dsDnA Detected by Enzyme immunoassays and indirect immunouorescence Assay (iFA)

reFerences

1. Brinkman K, Termaat R, Van den Brink H. The specificity of the anti-dsDNA ELISA. J Immunological Methods, 1991; 139: 91-100.2. Aarden LA, de Groot ER. and Feltkamp TEW. Immunology of DNA III. Crithidia luciliae, a simple substrate for the determination of anti-dsDNA with the immunouorescence technique. Ann NY Acad. Sci, 1975; 254: 505-15.

3. Stingl G. et al. An immunouorescence procedure for the demonstration of antibodies to native, double-stranded DNA and circulating DNA-AntiDNA complexes. Clin Immunol and Immunopathol, 1976; 6: 131-40.

4. Karamehic J, Subasic D, Gavrankapetanovic F, Zecevic L, Eminovic I, Memic S. et al. The incidence of antinuclear antibodies (ANA) detected by indirect immunouorescence assay (IFA) method. Med Arch, 2007; 61(1): 16-9.

5. Tonuttia E, Bassetti D, Piazza A, Visentini D, Poletto M, Bassetto F. et al. Diagnostic accuracy of ELISA methods as an alternative screening test to indirect immunouorescence for the detection of antinuclear

antibodies. Evaluation of ve commercial kits. Autoimmunity, 2004; 37(2): 171-6.6. Grripenberg M, Linder E, Kurki P, Engvall E. A solid phase enzyme-linked immunosorbent assay (ELISA) for the demonstration of antibodies against denaturated, single-stranded DNA in patient sera. Scand J Immunol, 1978; 7: 151-7.7. Lange A. Evaluation of the simultaneous estimation of anti-dsDNA and anti-ssDNA antibodies for clinical purposes. Clin Exp Immunol, 1978; 31: 472-81.

8. Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol, 2000; 53(6): 424-32.

9. Smeenk RJ, van den Brink HG, Brink-man K, Termaat RM, Barden JH, Swaak AJ. Anti-dsDNA: choice of assay in relation to clinical value. Rheumatol Int, 1991; 11(3): 101-7.

10. Villalta D, Romelli PB, Savina C, Bizzaro N, Tozzoli R, Tonutti E. et al. Anti-ds-DNA antibody avidity determination by a simple reliable ELISA method for SLE diagnosis and monitoring. Lupus, 2003; 12(1): 31-6.

11. Wigand R, Gottschalk R, Falkenbach A, Matthias T, Kaltwasser JP, Hoelzer D.

Detection of dsDNA antibodies in diagnosis of systemic lupus erythematosus comparative studies of diagnostic eectiveness of 3 ELISA methods with dierent antigens and a Crithidia luciliae immunouorescence test. Z Rheumathol, 1997; 56(2): 53-62.12. Nossent HC. and Rekvig OP. Is closer linkage between systemic lupus erythematosus and anti-double-stranded antibodies a desirable and attainable? Arthritis Rees Ther, 2005; 7(2): 85-7.

13. Smeenk R, Brinkman K, van den Brink H, Termaat RM, Berden J, Nossent H. et al. Antibodies to DNA in patients with systemic lupus erythematosus. Their role in the diagnosis, the follow-up and the pathogenesis of the disease. Clin Rheumathol, 1990; 9(1 Suppl. 1): 100-110.

14. Smeenk R, Swaak T. and Smeenk R. Clinical signicance of antibodies to double stranded DNA (dsDNA) for systemic lupus erythematosus (SLE). Clin Rheumathol, 1987; 6 Suppl. 1: 56-73.

15. Munoz LE, Gaipl US, Herrmann M. Predictive value of anti-dsDNA autoantibodies: importance of the assay. Autoimmun Rev, 2008; 7(8): 594-7.

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